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Impairment of the macroautophagy/autophagy-lysosomal pathway (ALP) can lead to amyloid plaque accumulation in Alzheimer disease (AD); however, the underlying mechanism remains unresolved. This study revealed a mechanism of ALP impairment mediated by gain-of-function of lysosomal TPCN2/TPC2 (two pore segment channel 2) and suggests a molecular target for AD intervention. Using mutant PSEN1/PS1 (presenilin 1)-expressing human neuroblastoma SH-SY5Y and familial AD fibroblasts collected from human patients, we showed lysosomal pH was increased in AD cells due to exaggerated TPCN2-mediated calcium (Ca2+) release which increases lysosomal pH and compromises ALP degradation. Genetic knockdown or pharmacological inhibition of the TPCN2 channel restored lysosomal Ca2+ homeostasis, acidity of the lysosome and ALP function. Furthermore, AD mice (5xFAD) that had received treatment with the TPCN2 inhibitor tetrandrine for 6 months or injection of AAV-shTpcn2 to knock down Tpcn2 showed reduction in amyloid plaques in cortical and hippocampal regions. These treatments also improved spine morphology and density and corrected cognitive deficits in 5xFAD mice assayed by immunohistochemistry, behavioral tests, and electrophysiological measurements, respectively. Taken together, these findings reveal a previously under-appreciated role of lysosomal TPCN2 in ALP impairment of AD. They also suggest targeting the lysosomal TPCN2 can serve as a therapeutic strategy for AD treatment in future drug development. Aβ: β-amyloid; AD: Alzheimer disease; AIF1/IBA1: allograft inflammatory factor 1; ALP: autophagy-lysosomal pathway; APP: amyloid beta precursor protein; ATP6V1B1/V-ATPase V1b1: ATPase H+ transporting V1 subunit B1; AVs: autophagy vacuoles; BAF: bafilomycin A1; CFC: contextual/cued fear conditioning assay; CHX: Ca2+/H+ exchanger; CTF-β: carboxy-terminal fragment derived from β-secretase; CTSD: cathepsin D; fAD: familial Alzheimer disease; GFAP: glial fibrillary acidic protein; LAMP1: lysosomal associated membrane protein 1; LTP: long‐term potentiation; MCOLN1/TRPML1: mucolipin 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPT: microtubule associated protein tau; MWM: Morris water maze; NFT: neurofibrillary tangles; PFC: prefrontal cortex; PSEN1: presenilin 1; SQSTM1/p62: sequestosome 1; TBS: theta burst stimulation; TEM: transmission electronic microscopy; TPCN2/TPC2: two pore segment channel 2; WT: wild-type; V-ATPase: vacuolar type H+-ATPase

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder. Amyloid-β (Aβ) and hyper-phosphorylated tau accumulation are accountable for the progressive neuronal loss and cognitive impairments usually observed in AD. Currently, medications for AD offer moderate symptomatic relief but fail to cure the disease; hence development of effective and safe drugs is urgently needed for AD treatment. In this study, we investigated a Chinese medicine (CM) formulation named NeuroDefend (ND), for reducing amyloid β (Aβ) and tau pathology in transgenic AD mice models. Regular oral administration of ND improved cognitive function and memory in 3XTg-AD and 5XFAD mice. In addition, ND reduced beta-amyloid precursor protein (APP), APP C-terminal fragments (CTF-β/α), Aβ and 4G8 positive Aβ burden in 3XTg-AD and 5XFAD mice. Furthermore, ND efficiently reduced the levels of insoluble phospho-tau protein aggregates and AT8 positive phospho tau neuron load in 3XTg-AD mice. Hence, ND could be a promising candidate for the treatment of AD in humans. Keywords: Alzheimer's disease, Aβ-plaque, Neurofibrillary tangles, Chinese medicine, NeuroDefend

The endosome-associated protein Rab5 is a central player in the molecular mechanisms leading to degeneration of basal forebrain cholinergic neurons (BFCN), a long-standing target for drug development. As p38α kinase is a Rab-5 activator, we hypothesized that inhibition of this kinase held potential as an approach to treat diseases associated with BFCN loss. Herein we report that treatment with an oral small molecule p38α kinase inhibitor reversed pathological disease progression in the basal forebrain in a mouse model that develops BFCN degeneration. Further, the preclinical results were successfully translated to the clinic, with improvement of clinical outcomes associated with cholinergic function in a clinical study in dementia with Lewy bodies (DLB), a disease in which BFCN dysfunction and degeneration is the primary driver of disease expression. The findings both advances a novel approach to treating DLB and validates the translational platform that provided the mechanistic rationale for advancing that approach.

Alzheimer’s disease (AD) is characterized by memory dysfunction, Aβ plaques together with phosphorylated tau-associated neurofibrillary tangles. Unfortunately, the present existing drugs for AD only offer mild symptomatic cure and have more side effects. As such, developments of effective, nontoxic drugs are immediately required for AD therapy. Present study demonstrates a novel role of Chinese medicine prescription Yuan-Hu Zhi Tong (YZT) in treating AD, and it has substantiated the in vivo effectiveness of YZT in two different transgenic mice models of AD, namely P301S tau and 3XTg-AD mice. Oral treatment of YZT significantly ameliorates motor dysfunction as well as promotes the clearance of aggregated tau in P301S tau mice. YZT improves the cognitive function and reduces the insoluble tau aggregates in 3XTg-AD mice model. Furthermore, YZT decreases the insoluble AT8 positive neuron load in both P301S tau and 3XTg-AD mice. Using microarray and the “Connectivity Map” analysis, we determined the YZT-induced changes in expression of signaling molecules and revealed the potential mechanism of action of YZT. YZT might regulate ubiquitin proteasomal system for the degradation of tau aggregates. The research results show that YZT is a potential drug candidate for the therapy of tau pathogenesis and memory decline in AD.

TFEB (transcription factor EB), which is a master regulator of autophagy and lysosome biogenesis, is considered to be a new therapeutic target for Parkinson&rsquo, s disease (PD). However, only several small-molecule TFEB activators have been discovered and their neuroprotective effects in PD are unclear. In this study, a curcumin derivative, named E4, was identified as a potent TFEB activator. Compound E4 promoted the translocation of TFEB from cytoplasm into nucleus, accompanied by enhanced autophagy and lysosomal biogenesis. Moreover, TFEB knockdown effectively attenuated E4-induced autophagy and lysosomal biogenesis. Mechanistically, E4-induced TFEB activation is mainly through AKT-MTORC1 inhibition. In the PD cell models, E4 promoted the degradation of &alpha, synuclein and protected against the cytotoxicity of MPP+ (1-methyl-4-phenylpyridinium ion) in neuronal cells. Overall, the TFEB activator E4 deserves further study in animal models of neurodegenerative diseases, including PD.

Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases in older individuals with specific neuropsychiatric symptoms. It is a proteinopathy, pathologically characterized by the presence of misfolded protein (Aβ and Tau) aggregates in the brain, causing progressive dementia. Increasing studies have provided evidence that the defect in protein-degrading systems, especially the autophagy-lysosome pathway (ALP), plays an important role in the pathogenesis of AD. Recent studies have demonstrated that AD-associated protein aggregates can be selectively recognized by some receptors and then be degraded by ALP, a process termed aggrephagy. In this study, we reviewed the role of aggrephagy in AD development and discussed the strategy of promoting aggrephagy using small molecules for the treatment of AD.

Collective evidences have indicated that intracellular accumulation of hyperphosphorylated tau forms neurofibrillary tangles in the brain, which impairs memory, cognition and affects social activities in Alzheimer's disease (AD).To investigate the tau-reducing, and memory-enhancing properties of protopine (PRO), a natural alkaloid isolated from Chinese herbal medicine Corydalis yanhusuo (Yanhusuo in Chinese).By using Histone deacetylase 6 (HDAC6) profiling and immunoprecipitation assays, we assessed that PRO mediated the heat shock protein 90 (HSP90) chaperonic activities for the degradation of pathological tau in AD cell culture models. To study the efficacy of PRO in vivo, we employed 3xTg-AD and P301S tau mice models.Liquid chromatography/quadrupole time-of-flight mass spectrometry was used to analyze the pharmacokinetic profile of PRO. Seven-month-old 3xTg-AD mice and 1.5-month-old P301S mice were administered PRO (1 and 2.5 mg/kg) orally every day. Morris water maze, contextual fear conditioning and rotarod assays were applied for studying memory functions. Sarkosyl differential centrifugation was used to analyze soluble and insoluble tau. Immunohistochemical analysis were performed to determine tau deposits in AD mice's brain sections. Molecular docking, binding affinity studies and primary cell culture studies were performed to demonstrate the mechanism of action of PRO in silico and in vitro.Our pharmacokinetic profiling demonstrated that PRO significantly entered the brain at a concentration of 289.47 ng/g, and specifically attenuated tau pathology, improved learning and memory functions in both 3xTg-AD and P301S mice. Docking, binding affinity studies, and fluorometric assays demonstrated that PRO directly bound to the catalytic domain 1 (CD1) of HDAC6 and down-regulated its activity. In primary cortical neurons, PRO enhanced acetylation of α-tubulin, indicating HDAC6 inhibition. Meanwhile, PRO promoted the ubiquitination of tau and recruited heat shock protein 70 (HSP70) and heat shock cognate complex 71 (HSC70) for the degradation of pathological tau via the ubiquitin-proteasomal system (UPS).We identified PRO as a natural HDAC6 inhibitor that attenuated tau pathology and improved memory dysfunctions in AD mice. The findings from this study provides a strong justification for future clinical development of plant-derived protopine as a novel agent for the treatment of tau-related neurodegenerative diseases.

Accumulating studies have suggested that targeting transcription factor EB (TFEB), an essential regulator of autophagy‐lysosomal pathway (ALP), is promising for the treatment of neurodegenerative disorders, including Alzheimer's disease (AD). However, potent and specific small molecule TFEB activators are not available at present. Previously, we identified a novel TFEB activator named curcumin analog C1 which directly binds to and activates TFEB. In this study, we systematically investigated the efficacy of curcumin analog C1 in three AD animal models that represent beta‐amyloid precursor protein (APP) pathology (5xFAD mice), tauopathy (P301S mice) and the APP/Tau combined pathology (3xTg‐AD mice). We found that C1 efficiently activated TFEB, enhanced autophagy and lysosomal activity, and reduced APP, APP C‐terminal fragments (CTF‐β/α), β‐amyloid peptides and Tau aggregates in these models accompanied by improved synaptic and cognitive function. Knockdown of TFEB and inhibition of lysosomal activity significantly inhibited the effects of C1 on APP and Tau degradation in vitro. In summary, curcumin analog C1 is a potent TFEB activator with promise for the prevention or treatment of AD., A small molecule activator of transcription factor EB (TFEB) promotes the degradation of beta‐amyloid precursor protein (APP) fragments, β‐amyloid peptides (Aβ), and phosphorylated MAPT/Tau aggregates in three transgenic mice models of Alzheimer's disease (P301S, 5xFAD, and 3xTg) and prevents memory impairments.

The autophagy-lysosome pathway (ALP) is a primary means by which damaged organelles and long-lived proteins are removed from cells and their components recycled. Impairment of the ALP has been found to be linked to the pathogenesis of Parkinson's disease (PD), a chronic neurodegenerative disorder characterized by the accumulation of protein aggregates and loss of dopaminergic neurons in the midbrain. In recent years, some active compounds derived from plants have been found to regulate the ALP and to exert neuroprotective effects in experimental models of PD, raising the possibility that autophagy enhancement may be an effective therapeutic strategy in PD treatment. In this review, we summarize recent findings of natural products that enhance ALP and thereby protect against PD. Research articles were retrieved from PubMed using relevant keywords in combination. Papers related to the topic were identified, and then the reliability of the experiments was assessed in terms of methodology. The results suggest that targeting the ALP with natural products is a promising strategy for PD treatment. However, risk of bias exists in some studies due to the defective methodology. Rigorous experimental design following the guidelines of autophagy assays, molecular target identification and in vivo efficacy evaluation is critical for the development of ALP enhancers for PD treatment in future studies. Copyright © 2017 John Wiley & Sons, Ltd.

Alzheimer’s disease (AD) is a degenerative disorder typified by progressive deterioration of memory and the appearance of β-amyloid peptide (Aβ)-rich senile plaques. Recently we have identified a novel function of a patented formulation of modified Huanglian-Jie-Tu-Tang (HLJDT-M), a Chinese herbal medicine, in treating AD in in vitro studies (US patent No. 9,375,457). HLJDT-M is a formulation composed of Rhizoma Coptitis, Cortex Phellodendri and Fructus Gardeniae without Radix Scutellariae. Here, we assessed the efficacy of HLJDT-M on a triple transgenic mouse model of AD (3XTg-AD). Oral administration of HLJDT-M ameliorated the cognitive dysfunction of 3XTg-AD mice and lessened the plaque burden. In addition, biochemical assays revealed a significant decrease in levels of detergent-soluble and acid-soluble Aβ via decreasing the levels of full length amyloid-β precursor protein (FL-APP) and C-terminal fragments of APP (CTFs) in brain lysates of HLJDT-M-treated mice. HLJDT-M treatment also significantly reduced the levels of FL-APP and CTFs in N2a/SweAPP cells. In contrast, treatment using the classical formula HLJDT did not reduce the memory impairment of 3XTg-AD mice and, rather, increased the Aβ/Fl-APP/CTFs in both animal and cell culture studies. Altogether, our study indicates that HLJDT-M is a promising herbal formulation to prevent and/or cure AD.

The present study explains the neuroprotective ability of bioactive fractions of Annona reticulata bark (ARB) and Ziziphus jujuba root bark (ZJ) along with insulin against diabetic neuropathy. By using different solvents of increasing polarity ARB and ZJ were undergone for bioactive guided fractionation. The neuroprotective ability of the all the plant fractions were tested against H2O2 induced toxicity in SHSY5Y neuroblastoma cell lines and DRG neuronal cells. Among all the fractions tested, the methanol extract of ARB and ZJ (ARBME and ZJME) and its water fractions (ARBWF and ZJWF) exhibited significant neuroprotection against H2O2 induced toxicity in SHSY5Y cells and DRG neuronal cells. Further both the active fractions were tested against streptozotocin (55 mg/kg i.p.) induced diabetic neuropathy in male Wistar rats. Body weight changes, blood glucose levels and pain threshold through hot plate, tail immersion, cold plate and Randall-Sillitto methods were measured throughout the study at weekly interval. After completion of the drug treatment period, all the animals were sacrificed to measure the sciatic nerve lipid peroxidation, antioxidative enzyme levels (SOD, catalase, and GSH) and cytokine levels (IL-1β, IL-6, IL-10, TNF-α, iNOS, and NFκB) through ELISA and western blotting analysis. Results of this study explain that ARBME, ZJME, ARBWF, and ZJWF along with insulin potentially attenuate the thermal, mechanical hyperalgesia and cold allodynia in diabetic neuropathic rats, where insulin treatment alone failed to diminish the same. Reduction of sciatic nerve oxidative stress, NF-κB and iNOS mediated inflammatory cascade and normalization of abnormal cytokine release confirms the possible mechanism of action. The present study confirms the neuroprotective ability of ARB and ZJ against painful diabetic neuropathy through inhibiting oxidative stress and NF-κB inflammatory cascade.

The autophagy-lysosome pathway (ALP) is a primary means by which damaged organelles and long-lived proteins are removed from cells and their components recycled. Impairment of the ALP has been found to be linked to the pathogenesis of Parkinson's disease (PD), a chronic neurodegenerative disorder characterized by the accumulation of protein aggregates and loss of dopaminergic neurons in the midbrain. In recent years, some active compounds derived from plants have been found to regulate the ALP and to exert neuroprotective effects in experimental models of PD, raising the possibility that autophagy enhancement may be an effective therapeutic strategy in PD treatment. In this review, we summarize recent findings of natural products that enhance ALP and thereby protect against PD. Research articles were retrieved from PubMed using relevant keywords in combination. Papers related to the topic were identified, and then the reliability of the experiments was assessed in terms of methodology. The results suggest that targeting the ALP with natural products is a promising strategy for PD treatment. However, risk of bias exists in some studies due to the defective methodology. Rigorous experimental design following the guidelines of autophagy assays, molecular target identification and in vivo efficacy evaluation is critical for the development of ALP enhancers for PD treatment in future studies. Copyright © 2017 John WileySons, Ltd.

The present study explains the neuroprotective ability of bioactive fractions of Annona reticulata bark (ARB) and Ziziphus jujuba root bark (ZJ) along with insulin against diabetic neuropathy. By using different solvents of increasing polarity ARB and ZJ were undergone for bioactive guided fractionation. The neuroprotective ability of the all the plant fractions were tested against H2O2 induced toxicity in SHSY5Y neuroblastoma cell lines and DRG neuronal cells. Among all the fractions tested, the methanol extract of ARB and ZJ (ARBME and ZJME) and its water fractions (ARBWF and ZJWF) exhibited significant neuroprotection against H2O2 induced toxicity in SHSY5Y cells and DRG neuronal cells. Further both the active fractions were tested against streptozotocin (55 mg/kg i.p.) induced diabetic neuropathy in male Wistar rats. Body weight changes, blood glucose levels and pain threshold through hot plate, tail immersion, cold plate and Randall-Sillitto methods were measured throughout the study at weekly interval. After completion of the drug treatment period, all the animals were sacrificed to measure the sciatic nerve lipid peroxidation, antioxidative enzyme levels (SOD, catalase, and GSH) and cytokine levels (IL-1β, IL-6, IL-10, TNF-α, iNOS, and NFκB) through ELISA and western blotting analysis. Results of this study explain that ARBME, ZJME, ARBWF, and ZJWF along with insulin potentially attenuate the thermal, mechanical hyperalgesia and cold allodynia in diabetic neuropathic rats, where insulin treatment alone failed to diminish the same. Reduction of sciatic nerve oxidative stress, NF-κB and iNOS mediated inflammatory cascade and normalization of abnormal cytokine release confirms the possible mechanism of action. The present study confirms the neuroprotective ability of ARB and ZJ against painful diabetic neuropathy through inhibiting oxidative stress and NF-κB inflammatory cascade.

Hippocampal neurogenesis plays a critical role in the formation of new neurons during learning and memory development. Attenuation of neurogenesis in the brain is one of the primary causes of dementia in Alzheimer’s disease (AD), and, conversely, modulating the process of hippocampal neurogenesis benefit patients with AD. Traditional Chinese medicine (TCM), particularly herbal medicine, has been in use for thousands of years in Asia and many regions of the world for the treatment of cancer, cardiovascular diseases and neurodegenerative diseases. In this review, we summarize the role of neurotrophic factors, signal transducing factors, epigenetic modulators and neurotransmitters in neurogenesis, and we also discuss the functions of several Chinese herbs and their active molecules in activating multiple pathways involved in neurogenesis. TCM herbs target pathways such as Notch, Wnt, Sonic Hedgehog and receptor tyrosine kinase pathway, leading to activation of a signaling cascade that ultimately enhances the transcription of several important genes necessary for neurogenesis. Given these pathway activating effects, the use of TCM herbs could be an effective therapeutic strategy for the treatment of AD.

Objective Generation and accumulation of the amyloid-β (Aβ) peptide after proteolytic processing of the full length amyloid precursor protein (FL-APP) by β-secretase (β-site APP cleaving enzyme or BACE1) and γ-secretase are the main causal factors of Alzheimer's disease (AD). Thus, inhibition of BACE1, a rate-limiting enzyme in the production of Aβ, is an attractive therapeutic approach for the treatment of AD. Recent studies suggest that salvianolic acid B (Sal B) is isolated from the radix of Salvia miltiorrhiza Bunge, a Chinese herbal medicine commonly used for the treatment of cardiovascular, cerebrovascular and liver diseases in China. Method In this study, we discovered that Sal B acted as a BACE1 modulator and reduced the level of secreted Aβ in two different Swedish APP (SwedAPP) mutant cell lines. Using N2a-mouse and H4- human neuroglioma cell lines expressing SwedAPP, it was demonstrated that Sal B significantly and dose-dependently decreased the generation of extracellular Aβ, soluble APPβ (by-product of APP cleaved by BACE1), and intracellular C-terminal fragment β from APP without influencing α-secretase and γ-secretase activity and the levels of FL-APP. In addition, using protein-docking, we determined the potential conformation of Sal B on BACE1 docking and revealed the interactions of Sal B with the BACE1 catalytic center. Results The docking provides a feasible explanation for the experimental results, especially in terms of the molecular basis of Sal B's action. Our results indicate that Sal B is a BACE1 inhibitor and, as such, is a promising candidate for the treatment of AD.

Herbal medicine is popularized worldwide due to its ability to cure the diseases with lesser or no side effects. North Eastern part of India comes under one of the world biodiversity hotspots which is very rich in traditional herbal medicine. Annona reticulata L. (Annonaceae) is one such plant used for the treatment of inflammatory diseases, liver ailments and diabetes by traditional healers. The present study was aimed to scientifically validate this folk knowledge and to develop an herbal remedy through evaluating bioactive guided fractions of A. reticulata (AR) bark against hepatotoxicity and inflammation using in vitro and in vivo models. Results of this study demonstrates that among all fractions of AR bark, methanol extract and its water fraction possess strong anti-oxidant ability and showed protection against CCl4 induced toxicity in HepG2 cell lines and rats. Both the fractions also exhibit dose dependent anti-inflammatory activity against carrageenan induced inflammation in rats. Water fraction showed potent response in the entire tests conducted than methanol extract, which states that polar components of the AR bark methanol extract were responsible for these activities. Further, from the experiments conducted to elucidate the mechanism of action, the results revealed that AR bark showed liver protection and anti-inflammatory response through inhibiting the oxidative stress and inflammatory cytokines.

Background Severe forms of non-alcoholic fatty liver disease (NAFLD) adversely affect the liver physiology and hence the pharmacokinetics of drugs. Here, we investigated the effect of NAFLD on the pharmacokinetics of rosiglitazone, an insulin sensitizer used in the treatment of type 2 diabetes. Methods Male C57BL/6 mice were divided into two groups. The first group (n=14) was fed with normal chow feed and the second group (n=14) was fed with 60% high-fat diet (HFD) and 40% high fructose liquid (HFL) for 60 days to induce NAFLD. The development of NAFLD was confirmed by histopathology, liver triglyceride levels and biochemical estimations, and used for pharmacokinetic investigations. Rosiglitazone was administered orally at 30 mg/kg dose. At predetermined time points, blood was collected and rosiglitazone concentrations were determined using LC/MS/MS. Plasma concentrations were subjected to non-compartmental analysis using Phoenix WinNonlin (6.3), and the area under the plasma concentration-time curve (AUC) was calculated by the linear-up log-down method. Results HFD and HFL diet successfully induced NAFLD in mice. Rosiglitazone pharmacokinetics in NAFLD animals were altered significantly as compared to healthy mice. Rosiglitazone exposure increased significantly in NAFLD mice (2.5-fold higher AUC than healthy mice). The rosiglitazone oral clearance was significantly lower and the mean plasma half-life was significantly longer in NAFLD mice as compared to healthy mice. Conclusions The NAFLD mouse model showed profound effects on rosiglitazone pharmacokinetics. The magnitude of change in rosiglitazone pharmacokinetics is similar to that observed in humans with moderate to severe liver disease. The present animal model can be utilized to study the NAFLD-induced changes in the pharmacokinetics of different drugs.